The present invention relates to a method of modifying polyphenol ozidase (PPO) activity in fruit and vegetables and to DNA sequences for use therein.
Browning of plant tissues often occurs following injury or damage and this generally results in spoilage of fruit and vegetables. Undesirable browning also occurs during processing of plant materials to produce food or other products. Steps are taken during transport, storage, and processing to prevent these browning reactions. Often this involves the use of chemicals such as sulphur dioxide but the use of these substances is likely to be restricted in the future due to concerns about their safety and consumer acceptance. For example, the US Food and Drug Administration banned the use of sulphite for most fresh fruit and vegetables in 1986. The production of fruit and vegetable varieties with an inherently low susceptibility to brown would remove the need for these chemical treatments.
Accordingly, it is an object of the present invention to overcome or at least alleviate one or more of the difficulties related to the prior art.
It will be understood that browning in plants is predominantly catalysed by the enzyme PPO. PPO is localised in the plastids of plant cells whereas the phenolic substrates of the enzyme are stored in the plant cell vacuole. This compartmentation prevents the browning reaction from occurring unless the plant cells are damaged and the enzyme and its substrates are mixed. If the amount of this enzyme could be decreased the susceptibility of the tissue to brown would be reduced.
PPO sequence information may be used to construct synthetic genes which genes may be transformed into plants to decrease expression of the normal PPO gene, thereby decreasing synthesis of the enzyme.
It will also be understood that in certain instances the browning reactions in plants are desirable, such as in the production of black tea, cocoa, coffee, black pepper, black olives, etc. In these instances it may be desirable tb increase the level of PPO to produce desired levels of browning or changes in flavour compounds.
The role of PPO in normal plant growth and development is not understood at present. There are a number of instances where increased levels of this enzyme are correlated with increased resistance to plant pathogens. It follow that genetic manipulation of plants to increase the level of PPO activity may confer useful resistance against pathogens and pests.
The grapevine PPO gene codes for an additional 103 amino acids upstream of the N-terminus of the mature protein. This region has the properties of a chloroplast transit peptide and is most likely responsible for targeting of the protein to be imported into the chloroplast and processed to produce the mature PPO protein. Transformation of plants with this gene may therefore result in correct targeting and maturation of the grapevine PPO in other species and result in accumulation of active grapevine PPO enzyme in the plastids of these tissues.
The terms “gene encoding PPO”, “gene coding for PPO” or “PPO gene” as used herein should be understood to refer to the PPO gene or a sequence substantially homologous therewith.